This invention relates to pulsed field gel electrophoresis of large DNA.
In the process of separating DNA molecules by electrophoresis, an electric field is applied across a gel to separate DNA molecules as they are moved by the field through the gel.
It is known to use the characteristics of the field established across the gel to control the electrophoresis for maximum separation. The fractionation of different molecular weight DNAs is presumably due to the sieving effect of the agarose gel matrix rather than differing electrophoresis mobilities of the DNAs as found in a free (completely liquid) medium.
In one prior art technique of electrophoresis that has been used for separating DNA, a static, unidirectional electric field is applied to a DNA sample resulting in the migration of the DNA molecules through the agarose.
This technique has a disadvantage in that it can successfully be applied to DNAs up to a few hundred kilobase. One embodiment of this technique can be successfully applied to DNAs up to 100 kilobase pairs (kb, about 60 million daltons, and another embodiment using low agarose gel concentration was described by Fangman, W.L. (1978), "Separation of very large DNA molecules by gel electrophoresis," Nucleic Acids Res. 5 (3):653-665, separated DNA molecules up to 750 kb. However, due to the inordinately long running times that are required when using low agarose concentrations gels and the inherent fragility of these gels, the method of Fangman is impractical for routine lab use.
Several other techniques are known to be successful in resolving large chromosome fragments sized (larger than 1 megabase) DNA molecules in agarose gels. These techniques are different forms of pulsed field gel electrophoresis (PFGE) which is the resolution of large sized DNA molecules by periodically changing the electric field pattern during electrophoresis. The changes in field pattern reorient the DNA molecules and the separating medium, thus improving DNA separation. In the prior art PFGE techniques, the pulse lengths are of sufficiently long duration to change the gross configuration of the DNA, being larger than one second in duration for the separation of large DNA. The changes in gross configuration are affected by the pulse duration and changes in direction and may vary from realigning direction of a substantially straight elongated strand to creating hooks or staircase-shaped strands.
Hooking and forking configurationsal changes in DNA during electrophoresis is described by Smith, S.B., Aldridge, P.k., and Calles, J.B. (1989), "Observation of Individual DNA Molecules Undergoing Gel Electrophoresis," Science 243:203-206. In this paper, Smith reported that DNA undergoes gross conformational changes during continuous (non-pulsed) electrophoresis. Gross changes are shown in a time scale of one second.
Historically, pulsed field electrophoresis was reported as early as 1959 by Schwalbe, M.I. (1959), "Pulsed Field Electrophoresis," International Conf. on Medical Electronics, pp. 603-604, Paris, in the separation of human plasma proteins using paper strip electrophoreseis but there are many other prior art embodiments. The technique described in that paper has not been used successfully to separate large DNA molecules.
In 1984, a pulsed field gel electrophoresis system (Pulsed Field Gradient Gel Electrophoresis), was reported in Schwartz, D.C. and Cantor, C.R. (1984), "Separation of Yeast Chromosome-sized DNAs by Pulsed Field Gradient Gel Electrophoresis," Cell 37 67-75; (1984) and in U.S. Pat. No. 4,473,452. This method was used in the separation of chromosome sized DNA.
In this method, an array of electrodes in a square submarine gel tank was used and two electric fields, one non-uniform and one uniform were pulsed in cycles of seconds or longer in two transverse directions in the plane of the gel; resulting in the resolution of DNA molecules with sizes from 30 to 2,000 kb. Schwartz, D.C. and Cantor, C.R. (1984), "Separation of Yeast Chromosome-sized DNAs by Pulsed Field Gradient Gel Electrophoresis," Cell 37:67-75; (1984) on page 68, and especially in table 1, describes 8 second perpendicular pulses of a uniform field.
A variation of the above was described in McPeek, F.D., Jr., Coyle-Morris, J.F., Gemmill, R.M. (1986), "Separation of Large DNA Molecules by Modified Pulsed Field Gradient Gel Electrophoresis," Anal. Biochemistry 156:274-285. A combination of non-uniform fields pulsing in the X- and Y-directions was examined. The results of this study showed that a cyclic pattern of non-uniform fields resulted in better DNA resolution in the PFGE System. More importantly, these studies demonstrated the significance of the pulsed field duration in times of seconds or longer in the resolution of different sized large DNA molecules. This led these researchers to conclude that the electric field switching time is a sensitive variable in OFAGE and probably all pulsed field gel electrophoresis techniques.
The field gradient techniques described by Schwartz, D.C. and Cantor, C.R. (1984) and by McPeek, F.D., Jr., Coyle-Morris, J.F., Gemmill, R.M. (1986) have a disadvantage in that the resulting pattern is non-linear and forms bent lanes, which seems to be due to the electric field gradient across the agarose gel. This gradient causes the direction of migration of the DNA molecules to vary depending on their location in the gel. The resulting bent lanes are of considerable consequence since this makes any lane-to-lane comparisons for molecular weight estimation difficult.
To alleviate this bent lane problem, the agarose gel has been oriented vertically so the electric field gradient is transverse, (across the thickness of the gel). This transverse alternating field is pulsed with pulse durations of seconds or more for separating large DNA. This method of electrophoresis (TAFE) eliminates the bent lanes but presets the value of the pulsed electric field angle to 115 degrees at the top of the gel to 165 degrees at the bottom. These methods are described in Gardiner, K., Laas W. and Patterson, D. (1986), "Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis," Somatic Cell Mol. Genet. 12:185-195, and in Gardiner, K. and Patterson, D. (1988), "Transverse Alternating Electrophoresis," Nature 331:371-372.
This method has a disadvantage in that the pulse duration is long and the electric field angle varies from 115 to 165 degrees along the gel although it is not adjustable. The field angle and pulse duration cause the DNA to move at an angle and the angle is a factor in PFGE which affects the separation resolution of DNA molecules. Thus, by causing the electric field angle to vary over a range of unadjustable values, the versatility of a technique related to a particular device is severely limited.
A simpler approach is described in Carle, G.F., Frank, M. and Olson, M.V. (1986), "Electrophoretic Separations of Large DNA Molecules by Periodic Inversion of the Electric Field," Science 232:65-68. In this approach, the electrophoretic separation of DNA molecules up to 700 kb is accomplished by using a method termed field inversion gel electrophoresis (FIGE). In FIGE, DNA molecules are subjected to a uniform electric field which is periodically inverted 180 degrees. Net forward migration of the DNA is achieved by differing the duration or the voltage of the forward and reversed fields.
FIGE has a disadvantage in that the resolution of DNA molecules larger than 200 kb is not as good as in the foregoing PFGE techniques. Although the problem of lane bending is eliminated, the rate of DNA migration is not monotonically related to size. Molecules of different sizes may have the same mobility. Thus, FIGE may not result in reliable DNA separation based on size.
The difficulty was explained by Sutherland et al. in Sutherland, J.C., Monteleone, D.C., Mugavero, J.H. and Trunk, J. (1987), "Unidirectional Pulsed-Field Electrophoresis of Single- and Double-stranded DNA in Agarose Gels: Analytical Expressions Relating Mobility and Molecular Length and Their Application in the Measurement of Strand Breaks," Anal. Biochemistry 162:511-520. This paper describes an attempt to solve this problem. They reexamined DNA agarose gel separation using unidirectional pulsed field electrophoresis where the electric field is pulsed in one direction without inversion. Although these authors show that DNA size is a function of mobility in their system, the upper DNA resolution limit is in the range of 400 kb.
In another prior art technique described by Chu and coworkers in Chu, G., Vollrath, D. and Davis, R.W. (1986), "Separation of Large DNA Molecules by Contour-clamped Homogeneous Electric Fields," Science 234:1582-1585, the electric field vector was examined and a conclusion was reached that the limitations of a non-uniform electric field could be overcome by applying a contoured-clamped homogeneous electric field (CHEF) which alternates between two orientations. He concluded that changing the electric field angle from 0 to 153 degrees improved separation of DNA up to 200 kb.
To apply a contoured-clamped homogeneous electric field (CHEF) which alternates between two orientations, the CHEF system uses a hexagonal tank with multiple electrodes, which effectively sets the value of the uniform electric field angle to 120 degrees and clamps each electrode to the appropriate potential. In the same article, Chu and his coworkers also report on a square tank using uniform and perpendicular fields.
Thus, with this system, Chu and coworkers demonstrated that the separation of large DNA molecules is a function of the electric field angle and electric field pulse duration. This apparatus has the disadvantage that the field angle cannot easily be varied. A way of varying field angle is descrived in Serwer in Serwer, P. (1987), "Gel Electrophoresis with Discontinuous Rotation of the Gel: An Alternative to Gel Electrophoresis with Changing Direction of the Electric Field," Electrophoresis 8:301-304. This paper describes the effect of changing the electric field angle by mechanically rotating the agarose gel, (Rotating gel electrophoresis, RGE).
CHEF and RGE have a disadvantage in that the pulse cycle times are longer than several seconds for separating large DNA, causing periodic and frequent changes in the gross configuration of the DNA, and possible reduction in the resolving ability of the system. In addition to the disadvantage of changes in the gross configuration of the DNA, unlike the CHEF system, RGE suffers from the rotational forces which can stress the agarose gel and also from mechanical complexity.
In still another embodiment of PFGE, described by Hood and fellow researchers in Birren, B.W., Lai, E., Clark, S.M., Hood, L. and Simon, M.I. (1988), "Optimized Conditions for Pulsed Field Gel Electrophoretic Separations of DNA," Nucleic Acids Res. 16:7563-7582 and Clark, S.M., Lai, E., Birren, B.W. and Hood, L. (1988), "A Novel Instrument for Separating Large DNA Molecules with Pulsed Homogeneous Electric Fields," Science 241:1203-1205, a programmable, autonomously controlled electrode gel electrophoresis (PACE) apparatus allows for the control of the electric field parameters. The PACE system includes a buffer tank with 24 independently regulated electrodes allowing the user to control pulse times and electric field angles. However, PACE also includes 24 high voltage amplifiers with 24 identical sets of digital-to-analog converter amplifiers in combination, all controlled by a personal computer.
The PACE apparatus, although versatile, has a disadvantage in that it is costly and its practical use in research labs appears to be limited.
The use of perpendicular pulses of electric fields which are repeated to form a reversing stairstep pattern is reported in Bancroft, I. and Walk, C.P. (1988), "Pulsed Homogenous Orthogonal Field Gel Electrophoresis (PHOGE)," Nucleic Acids Res. 15:7405-7418. One field pulse is parallel to the actual direction of migration and other pulses are either 90 degrees to the right or 90 degrees to the left of the direction of migration.
Another prior art technique of significance is a variation of PHOGE and also produces stairsteps from long duration pulses. This technique is described in Schwartz, D. C., and Koval, M. (1989), "Conformational dynamics of individual DNA molecules during gel electrophoresis," Nature 338:530-522. It reports the use of alternate perpendicular pulses of 6 to 8-second pulse pair periods, from 3 to 5 seconds for each of the two perpendicular pulse pairs (1/6 to 1/8 Hz).
With multiple pulse repeats at these low pulse frequencies, DNA bends in a multiple stairstep or staircase-like configuration with a bend each time the field alternates from one perpendicular direction to the other. This is as predicted in Schwartz, D..C. (1985), "Giga-Dalton Sized DNA Molecules," pp. 81-83, doctoral dissertation Columbia University (University Microfilms International).
After a longer period, 80 to 90 seconds, Schwartz and Koval reverse the polarity of one of the pulse fields so that the general trend of the staircase pattern alternates through an angle of 90 or 120 degrees, depending on the ratio of the pulse widths in the pulse pair period. The 80 to 90 second period corresponds to the pulse cycles used by previous workers. The staircase effect results from allowing sufficient time between pulses for gross changes in configuration (generation of the staircase pattern itself), which is what happens in the 8 second perpendicular pulse separation reported by Schwartz and Canter (1984) in the Cell article.
The use of uniform, pulsed perpendicular fields is also reported by Chu et al. and by Bancroft and Wolk (supra). Separation by slow (greater than 1 second) complexly pulsed electrophoresis as reported by Schwartz and Koval does not differ in principle from that described by Schwartz and Canter, Chu and especially by Bancroft and Wolk because all deal with perpendicular changes in field direction followed by enough time for new gross changes in DNA conformation to take place before the next field change. Bancroft and Wolk also use a similar directional change pattern for the electric field.
Each of the prior art pulsed field techniqes has the disadvantage of using a time duration for changing the field pattern that is in the order of a second or longer for separating large DNAs.
In the past, orthogonal pulses of duration too short to allow change in DNA configuration to take place were expected to appear as a vector sum, and be generally useless for separating DNA. This was predicted in Schwartz, D.C. (1985) "Giga-Dalton Sized DNA Molecules," p. 84, doctoral dissertation, Columbia University (University Microfilms International). The utility of such short pulses is a surprising result.